Previously, calculations of the lumen-exposed protonatable residue pKa values in PsbS from spinach were described in the literature. However, it has recently become clear that PsbS, although typical of higher plants and charophytes, can also provide photoprotection in green algae. Namely, the stress-induced expression of PsbS was recently shown for two green microalgae species: Chlamydomonas reinhardtii and Lobosphaera incisa. Therefore, we determined the amino acid sequence and modeled the three-dimensional structure of the PsbS from L. incisa, as well as calculated the pKa values of its lumen-exposed protonatable residues. Despite significant differences in amino acid sequence, proteins from L. incisa and Spinacia oleracea have similar three-dimensional structures. Along with the other differences, one of the two pH-sensing glutamates in PsbS from S. oleracea  has no analogue in L. incisa protein. Moreover, there are only four glutamate residues in the lumenal region of the L. incisa protein, while there are eight glutamates in S. oleracea. However, our calculations show that, despite the relative deficiency in protonatable residues, at least two residues of L. incisa PsbS can be considered probable pH sensors: Glu-87 and Lys-196.PsbS is one of the key photoprotective proteins, ensuring the tolerance of the photosynthetic apparatus (PSA) of a plant to abrupt changes in irradiance. Being a component of photosystem II, it provides the formation of quenching centers for excited states of chlorophyll in the photosynthetic antenna with an excess of light energy. The signal for “turning on” the photoprotective function of the protein is an excessive decrease in pH in the thylakoid lumen occurring when all the absorbed light energy  cannot be used for carbon assimilation. Hence, lumen-exposed protonatable amino acid residues that could serve as pH sensors are the essential components of PsbS-dependent photoprotection, and their pK The photosynthetic apparatus (PSA) of a plant provides plant growth and, at the same time, serves as a potential source of reactive oxygen species (ROS), damaging the PSA. This occurs in excess light, when the rate of electron transfer reactions limits the overall light phase of photosynthesis. As a result, low-potential reduced redox cofactors accumulate in the electron transport chain, and chlorophyll (Chl) excited state lifetime in photosynthetic antennas increases. Both of them (reduced redox cofactors and excited Chl) interact with molecular oxygen to form ROS ,2,3. ConPhyscomitrella patens possessing both photoprotective proteins, LhcsR and PsbS, the PsbS gene knockout leads to a remarkable decrease in NPQ [NPQ is typical for all the oxygenic phototrophs, including cyanobacteria, algae, and higher plants ,7. Severe in NPQ .Physcomitrella patens PsbS, the NMR and IR spectroscopic data revealed repositioning of H2 from the aqueous to the membrane phase and switching H3 from a loop segment into a helix caused by protonation of key glutamate residues [Suggestions about how PsbS performs its photoprotective function have changed significantly over time. Initially, it was assumed to serve as a direct quenching center due to the associated carotenoid molecules (zeaxanthin) ,15. But residues .Arabidopsis thaliana and the moss Physcomitrella patens, a set of mutants with replaced glutamate residues was obtained. In both species, two lumen-exposed glutamate residues were especially critical for the function of PsbS. When the three-dimensional structure of spinach PsbS became accessible, it also became possible to calculate the pKa values of all assumed pH-sensing groups [However, regardless of the mechanism of PsbS-dependent quenching, switching PsbS to the active state in response to lumen acidification remains the key process. This switching is obviously caused by the protonation of some lumen-exposed amino acid (AA) residues. While qE dependence on intrathylakoid pH has been numerously documented in the literature , proving groups .Chlamydomonas reinhardtii [Lobosphaera incisa [a values of its AA residues, which determine the lumenal pH range for NPQ activation. In this regard, we searched for transcripts encoding this protein in L. incisa, read its presumed amino acid sequence, simulated its three-dimensional structure and calculated the pKa values of its probable pH-sensing residues. In this article, we present the results of these calculations, as well as data on the amino acid sequence and the three-dimensional structure of the protein. We compare them with the data known to date on other organisms and analyze them with regard to structure versus function relations.As mentioned above, PsbS is typical for higher plants and Chara algae. In all other groups of algae, the same function  is pnhardtii ,30. Recea incisa , which mThe CDS of the gene encoding PsbS , as well as from a higher plant S. oleracea. There are 19 highly conservative sites in the protein structure. This is significantly less than what was revealed when comparing the PsbS sequences of higher plants (including the moss Physcomitrella patens), which were 130 (of ca. 200) fully conserved residues, arranged in compact or even continuous groups up to 23 amino acid (AA) residues in length [L. incisa and S. oleraca protein sequences reveals a less than 33% identity (68 AA out of 210/212 AA). Moreover, all the 68 AAs are mostly dispersed along the gene singly or in groups of 2 or 3 AAs, and only 16 AAs are concentrated in three relatively small groups  (n length . This presidues) . Most ofTo predict the spatial structure of a chloroplast protein based on its mRNA sequence, the chloroplast signal sequence at the N-terminus should be taken into account. The length of the chloroplast signal sequence varies from 13 to 146; most (>80%) chloroplast transit peptides consist of 30–80 residues . For theS. oleracea PsbS, despite the significant difference in their amino acid sequences (L. incisa–PsbS is slightly longer (by 1–1.5 turns) than that of S. oleracea (Thr-33—Gly-59), and the other transmembrane helices are almost the same in length: TM2, TM3, and TM4 are built of Ile-111—Leu-125, Lys-175—Thr-198, and Leu-215—Ala-234, respectively, which corresponds to TM2–TM4 alpha helical regions in S. oleracea–PsbS: Pro-79—Ile-93, Ser-139—Gly-163, and Asn-179—Asn-198. The TM1 and TM2, as well as TM3 and TM4, are interconnected by relatively short sections exposed in lumens, with a short (2–2.5 turns) alpha helix located between TM3 and TM4 and approximately parallel to the membrane in both proteins. Also in both proteins, the TM2 and TM3 are connected by a large (about 50 AA) stroma-exposed loop. The loop is highly mobile, which can be concluded from the X-ray structure of the S. oleracea–PsbS (PDB code 4RI2) where it is not resolved. Our data on MD simulation of L. incisa–PsbS are consistent with this conclusion.The predicted three-dimensional structure of the protein optimized using MD simulation is very close to that of the structure of equences . Both prL. incisa–PsbS contains 42 ionogenic residues, including 9 Arg, 12 Lys, 4 Tyr, 10 Glu, and 7 Asp residues . Neither His nor Cys residues, which could also be considered candidates for the role of pH sensors, are present in the mature protein. Nine of them are located at the lumenal side of the protein: glutamates 101, 195, 87, and 214, aspartate 105, tyrosine 219, lysines 92, 196, and 201 , -159, and -180 in PsbS from S. oleracea  of this protein region (ε) is much higher. The stroma-exposed region of the protein is much larger than the lumen-exposed one; therefore, the contribution of polar (including ionic) AA residues to the overall ε of this protein region is more significant. This results in a high ε (~20) of the stroma-exposed region. On the contrary, hydrophilic loops connecting the transmembrane helices in the lumen-exposed region are relatively small, and even the eight ionic residues mentioned above cannot provide a significant up-shift in the ε values. We assume that such a design of the dielectric properties of the protein is not accidental; rather, it is important for the functions of the protein. The low ε of the lumen-exposed region is a prerequisite to providing (i) strong local electric fields and (ii) high values of the dielectric response of the medium; both factors ensure a larger shift in the pKa of the lumen-exposed ionic AA residues toward their biologically relevant values .As expected, inside the hydrophobic part of the membrane, mostly hydrophobic AA residues are located, which leads to the lowest dielectric permittivity  and Lys-196 (pKa 5.81 ± 0.02). As for Lys-196, in both chains, we observed its sidechain somewhat submerged in the protein, which should contribute to discharging of the residue, i.e., to the down-shift in its pKa value toward the neutral pH range. This seems to indicate its physiological role as a pH sensor. However, in one of the chains, we observed a permanently protonated state of Lys-196 stabilized by hydrogen bonds (H-bonds) formed by hydrogen atoms of its terminal amino group with carbonyl oxygen atoms of the backbone . As for Glu-87, in the other chain it also revealed a higher pKa value compared with the other glutamates, although it was insufficient to provide its physiological role (~5.0).According to our calculations, there is a slight asymmetry in the pKa toward the physiologically irrelevant alkaline range. At least in one of the chains, we observed an H-bond formed by a Glu-195 carboxyl proton with one of the carboxyl oxygen atoms of Glu-101. This, in turn, stabilizes Glu-101 in the deprotonated state in the neutral and slightly acidic pH region. Nevertheless, in the other chain, Glu-101 did not form this H-bond and manifested a pKa value close to 5.0.On the contrary, Glu-195 in both chains forms an H-bond , which stabilizes its protonated state and up-shifts pKa shifts due to the dielectric response energy should be negligible. Moreover, in one of the chains, we observed an additional stabilization of the deprotonated state of Asp-105 due to two H-bonds formed by its carboxyl oxygen atoms with the backbone hydrogen atom of Gly-107 and one of the hydrogen atoms of Pro-106. All this makes Glu-214 and Asp-105 unlikely candidates for the role of physiologically significant pH sensors.As for Glu-214 and the aspartate residue Asp-105, they are located almost on the surface of the protein, so their pKa significantly. Thus, Tyr-219 is submerged into the protein, which causes a slight stabilization of its neutral (protonated) form. Moreover, the hydroxyl proton forms (at least part of the time) a hydrogen bond with the backbone oxygen of Leu-88, which ensures additional stabilization of the protonated Tyr-219.As for tyrosine and two other lysine residues (Lys-92 and -201), there do not seem to be any effects able to downshift their pKS. oleracea, the lumen-exposed protonatable (presumably glutamate) residues tend to form a much more remarkable pH-sensitive cluster compared with that of L. incisa’s PsbS, as was shown for the first time by [S. oleracea, reveal four glutamate residues  with the pKa values upshifted above 6.0. We obtained pKa values for this cluster of 6.2, 6.3, 6.4, and 7.2, which are close to those calculated by [a values below 6.0, the highest of them being 5.1 for Glu-76 and 4.7 for Glu-69 , and an error of a graphical assessment of pKa does not exceed ±0.03 pH units. To assess the error coming from the uncertainty of parameters in our model, we varied some parameters and calculated the corresponding pKa values. In most of the calculations, the membrane was considered to be dielectrically inhomogeneous, with ε = 2.0 for its inner region containing nonpolar fatty acid residues and ε = 20.0 for the outer regions containing hydrophilic heads. We also tested ε = 40.0 for the outer regions, which is close to the dielectric permittivity of glycerol [a values of two lumen-exposed residues by 0.06–0.08 pH units; for most of the lumen-exposed residues, the changes were in the region of 0.01–0.02 pH units. The effect of taking ε = 2.5 instead of 2.0 for the inner membrane region was less than 0.02–0.03 pH units for all these residues. The variations in the ionic strength of the solution in the range of 0.05–0.2 M exerted an effect of the same order of magnitude.To draw reliable conclusions regarding the calculated pKglycerol . This cha values should be made by fluctuations in the atomic structure of the protein. To account for this contribution, we calculated the fluctuations in the two most significant components determining the pKa shift in the protein: the change in the dielectric response energy and the effect of the pre-existing electric field created by partial charges of neighboring atoms (presumably by the atoms of the backbone), excluding the atoms of protonatable groups. The third component, i.e., determined by the dielectric interaction between the protonatable groups, only accounted for pairs interacting with energy that was not less than 0.5 pH units. Usually, each ionogenic residue interacted significantly with no more than two neighbors; for most pairs, the interaction energy was relatively small, not exceeding 0.1 pH units. All the components were calculated along the last 200 ns section of the MD trajectory with a 1 ns step. The sum of all the components varied with a standard deviation (SD) ranging from 0.38 pH units for Glu-214 to 2.2 pH units for Glu-195. This is not surprising since Glu-214 is the most water-exposed surface residue; hence, it is influenced by the protein environment to the least extent. A similar situation takes place in the case of the other most “external” residue, Asp-105 (SD = 0.52). Conversely, the residue Glu-195 interacts with the protein most strongly, mainly due to the H-bonds formed with the surrounding. The fluctuations in this large value are also large. The median value of the SD of fluctuations determining the pKa shift of all the lumen-exposed ionic residues is 0.85.It is reasonable to assume that the most significant contribution to the error in determining the pKa variations since proton exchange occurs rather slowly compared to the structure fluctuation times studied here. Thus, typical site-specific protonation and deprotonation rate constants, kon and koff, vary in the ranges (0.6–300) × 109 M−1 s−1  and (0.1–3) × 106 s−1 , respectively [a value.Note, however, that strictly speaking, these variations cannot be considered pKectively . Therefoa shift, we obtained pKa ≈ 6.5 ± 0.1 for Glu-87 and pKa ≈ 6.3 ± 0.15 for Lys-196.Taking into account 200 ns fluctuations of the electric interaction-induced pKC. reinhardtii [The PsbS protein was previously considered typical for the evolutionary branch of higher plants . Recent studies, however, significantly lengthen its history. Initially, the PsbS gene was found in the Chlorophyta genome ,36,37. Onhardtii ,30. The nhardtii  demonstrL. incisa–PsbS and its most studied homologue from S. oleracea, a higher plant. This indicates the functional meaning of this degree of lumen acidification, which is crucial for NPQ induction even in such distant photosynthetic organisms. On the other hand, the AA sequence of the L. incisa–PsbS is strikingly different from that of S. oleracea–PsbS and even of the PsbS from the other green algal species; moreover, this difference is observed along the entire polypeptide chain, including the regions with lumen-exposed ionic residues essential for PsbS functioning. e.g., L. incisa–PsbS contains half as many glutamate residues in the lumenal region of the protein as S. oleracea–PsbS (4 instead of 8); moreover, it lacks one of the two pH-sensing glutamate residues  typical for both S. oleracea–PsbS (Glu-173) and A. thaliana–PsbS (Glu-226). Although there is an additional aspartate residue (Asp-105) in L. incisa–PsbS, it does not save the situation since it is located in a loop strongly protruding into the aqueous phase (this makes a noticeable protein-dependent pKa shift impossible). Perhaps that is why Lys-196  turns out to be essential in pH sensing. Its location is deep enough inside the protein; moreover, near the anionic residues Glu-195 and Glu-101 is a good prerequisite for a significant pKa shift toward a neutral or slightly acidic region. This comparison of two proteins is a good example of how functional properties rather than structure can be conserved during evolution.Our data show that there is no significant difference in the pH sensitivity between a values of the same residues in two monomers. Note that we obtained pKa values that fit into the physiologically important range only for one of the monomers. We realize that this may be due to an insufficient simulation time. However, on the other hand, recognition of the significance of simulation time can be interpreted as the existence of several relatively long-lived states of a protein that differ in their pH sensitivity. We believe that this assumption is in line with modern ideas about how the functional properties of proteins are related to their thermal fluctuations; e.g., fluorescence intermittency (blinking) originating from fluctuations around several discrete protein states of photosynthetic antenna and underlying the environmental control over light-harvesting/thermal dissipation is a vivid example of this relation [In our study, we considered (homo)dimer of PsbS; however, we revealed a certain asymmetry: the calculation gave different  pKrelation .a values obtained for the PsbS protein in the literature to date [a values of PsbS residues obtained in model systems, including those resulting from MD simulation. One possible interpretation may be the conclusion that such data represent an artifact, while some other residues play a role in switching PsbS to the quenching state in vivo—e.g., those with pKa values shifting to the physiologically significant range upon contact with Zea and/or components of antenna. However, this assumption does not seem to agree well with the data on mutagenesis of residues Glu122 and Glu226 in A. thaliana–PsbS [S. oleracea–PsbS [Despite the reasonable range of pK to date , as well to date ,39 along to date ,40. Howe to date ,41, and ana–PsbS  equivalecea–PsbS . AnotherNevertheless, the study of the pH-dependent properties of the PsbS protein even in such model systems makes a lot of sense since only a comparison of theoretical and experimental data, both model and in vivo, can make it possible to find out the structural and functional relationships in this protein and in the entire photoprotective system of a plant.Lobosphaera incisa IPPAS C-2047  was cultivated as described earlier [L. incisa DNA homologous to the psbS gene of vascular plants  and RNA was isolated according to the manufacturer’s protocol. Single-stranded cDNA was synthesized using MMLV RT kit with Mint reverse transcriptase  and random or Oligo(dT)15 primers according to the manufacturer’s protocol; 3200 ng of RNA was used for cDNA synthesis. The obtained cDNA was stored at −20 °C.Total RNA was extracted from the cell sample using RNeasy Mini kit . Cell pellet frozen in liquid nitrogen (~100 mg) was homogenized in a porcelain mortar. Plant RNA Isolation Aid  was added to the homogenate along with RLT lysis buffer, then clarified by centrifugation  [Chlamydomonas reinhardtii or Volvox carteri, the only available green algal species, were indicated as target organisms). Four start codons closest to the 5′-end and three stop codons closest to the 3′-end were chosen. The sequence ending with a start codon was used to design a 5′-end primer . Subsequently, Psbs-fwd (#1) with one of the 3′-end primers (##3–5) was used in PCR to amplify the 3′-end of cDNA . To amplify the 5′-end of cDNA, Psbs-rev (#2) with one of the 5′-end primers (##6–9) was used in PCR. Based on the CDS prediction, the probable lengths of PCR products were predicted for each primer pair  . Severalmer pair .15 primers were used in PCR. The former was used in PCR with each of seven primer pairs (#1 + #(3 to 5) and #2 + #(6 to 9)). The latter was additionally used in PCR with #1+ Oligo(dT)15 primer pair. The analysis of PCR products obtained with each of the primer pairs (except for #1 + Oligo(dT)15, #2 + 8, and #2 + #9) using agarose gel electrophoresis revealed the only band corresponding to the predicted length. Products with the maximum length  were Sanger sequenced using Evrogen facilities . These two sequences corresponded to two semi-halves of the gene (including some fragments of 5′- and 3′-UTR) and had a common section of 146 bp length; they were combined into one sequence with a total length of 1024 bp (presumably containing the entire sequence of PsbS gene) by alignment along the common section. In the combined sequence, the location of CDS was predicted using Augustus online software (see Both cDNA samples generated from an RNA template with random or Oligo(dT)ware see .https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 3 February 2023) was used.For global alignment, ClustalW Multiple Alignment, implemented in Sequence Alignment Editor BioEdit 7.2.5 , was usehttps://www.ncbi.nlm.nih.gov/genbank/ (accessed on 10 July 2023) for Haematococcus lacustris (ID: AUI41105.1) or Phytozome database https://phytozome-next.jgi.doe.gov/ (accessed on 10 July 2023) for Botryococcus braunii (ID: Bobra.139_1s0009.1), C. reinhardtii (ID: Cre01.g016600_4532.1), Micromonas pusilla (ID: MicpuC2.EuGene.0000110272), as well as for vascular plant Spinacia oleracea (ID: Spov3_chr5.00161).The sequences of PsbS protein from the green algae were taken from GenBank, http://wormweb.org/exonintron (accessed on 10 July 2023). To visualize results of alignment, online service ESPript, https://espript.ibcp.fr (accessed on 30 September 2023) was used [The exon–intron structure based on the alignment of PsbS mRNA and contig GenBank OR604506 sequences is visualized using the online service was used .https://services.healthtech.dtu.dk/service.php?TargetP-2.0 (accessed on 6 May 2023) employing machine learning methods [http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index (accessed on 8 May 2023) in the “Intensive mode” based on the protein homology to known structures as well as on ab initio folding simulation [First, the chloroplast signal sequence at the N-terminus was predicted using online service TargetP-2.0,  methods . The premulation .https://charmm-gui.org/ (accessed on 4 June 2023) [3 size contained a protein, 149 lipid molecules, 6486 water molecules (TIP3P model) and 195 ions (K+ and Cl−). The input files for GROMACS were generated by the special module [The obtained structure was used for building an input structure for molecular dynamics (MD) simulation by CHARMM-GUI Simulation Input Generator ne 2023) . A bilayne 2023) . The prol module .a values . For the resulting dimer embedded in the lipid membrane, MD simulation was performed again for 0.2 microseconds. Thus, the total MD simulation time was 1.4 microseconds. The resulting structure was used to calculate the pKS. oleracea (PDB code 4RI2) with similar parameters of the system: 75 × 75 × 87 Å3 cell containing 130 lipid molecules, 8863 water molecules (TIP3P model), and 188 ions (K+ and 30 Cl−).The MD simulation was also performed for PsbS dimer from a value of each ionogenic residue in PsbS protein, we estimated its shift upon the residue transfer from water solution into the definite site in the protein within the framework of the semi-continuum dielectric model. Three main components of the transfer energy were considered: (i) the change in the dielectric response energy  and (ii) the effect of the pre-existing electric field created at a given site within the protein by permanent partial charges of neighboring atoms. After that, (iii) the dielectric interaction between the protonatable groups was accounted for.To calculate the pKε) within a protein, we employed correlation between local concentration of polar groups in a definite protein volume, on the one hand, and the static dielectric permittivity of the same volume, on the other hand. Previously, we thoroughly investigated this correlation using a well-studied bacterial photosynthetic reaction center, a protein complex for which the experimental data on the permittivity of its various domains are available. Following the algorithm we developed [ε value determined in the work [ε distribution and then grouped the regions with similar ε values into four layers, each of which was assigned an average ε value: 3.0, 5.0, 10.0, or 20.0. The obtained “discrete” ε distribution was used to calculate the change in the dielectric response energy upon residue transfer from water into protein and the dielectric interaction between the protonatable groups. Note that the obtained ε is static dielectric permittivity (εs), i.e., it accounts for both the inertial and electronic parts of the medium polarization.In these calculations, we used the approach developed in our previous works ,54 and beveloped , in the the work , we calcεs = 2.0 in most of the experiments and εs = 2.5 while assessing the probable error resulting from variations in this parameter. For the outer regions containing hydrophilic heads, we used the experimental data obtained for glycerol [εs ≈ 40.0, and the latter gave the range εs ≈ 26–70 for different forms of starch. In our calculations, we used εs ≈ 40.0 or 20.0 for this outer membrane region to assess the probable error of the dielectric model. The protein embedded in the membrane was surrounded by a continuous medium (aqueous solution) with ε = 81 and ionic strength of 0.2 M.In electrostatic calculations, the protein was surrounded by a bilayer PG membrane in a configuration obtained in MD simulation. The membrane was considered to be dielectrically inhomogeneous. For the inner region of the membrane formed by fatty acid residues, we used glycerol  and starglycerol . The forTo calculate the electric field and dielectric response energies, the Poisson–Boltzmann equation (PBE) was solved using DelPhi  V. 4 Rela values used as input data for Karlsberg were calculated as the sum of two components of the transfer energy of a residue: the shifts caused by the change in the dielectric response energy (the sign depends on the charge of protonated residue) and by the intraprotein electric field.The protonation states of ionizable residues  were obtained by the Monte Carlo sampling method using the program Karlsberg version 1.0.1 ,59. The a values, are expected to play a crucial role.PsbS is a universal photoprotective protein among higher plants. Recent studies (including our own) have shown that the history of its functioning in phototrophs began at least with green algae. Given the different habitat as well as the fact that LhcsR-dependent photoprotection (absent in vascular plants) is active in green algae, one can expect a significant difference in the pattern of activation of the PsbS-dependent photoprotective pathway. This difference should correlate with the structure and physico-chemical properties of the protein. Since the activation of photoprotection is pH-dependent, the appropriate molecular tools, i.e., the set of protonatable residues of a protein and their pKL. incisa–PsbS and S. oleracea–PsbS , including the regions with lumen-exposed ionic residues. L. incisa–PsbS contains only four glutamate residues in the lumenal region of the protein (compared with eight in S. oleracea–PsbS). Moreover, one of the two pH-sensing glutamate residues typical for both S. oleracea–PsbS (Glu-173) and A. thaliana–PsbS (Glu-226) is absent in L. incisa–PsbS. Nevertheless, the overall three-dimensional structure of the protein is very close to that of its homologues. Further, two of the protonatable lumenal-exposed residues, Glu-87 and Lys-196, are predicted to possess pKa values in the slightly acidic region essential for triggering PsbS into protective mode. This may indicate that the PsbS-dependent photoprotective mechanism and the conditions for switching it on do not differ significantly in green algae and higher plants. However, this issue requires future thorough research.Our study revealed a significant difference between the AA sequences of


The supply of fish oil, which provides long-chain polyunsaturated fatty acids (LC-PUFA) but is mostly wild-caught, is stagnating, resulting in limited availability and high prices. Therefore, many alternative lipid sources, mainly vegetable oils and rendered animal fats, are increasingly being applied in fish feeds. However, these lipid sources do not contain LC-PUFA, and therefore lower the fillet quality in terms of the LC-PUFA content. Feeding strategies such as alternate feeding between fish oil- and alternative oils-based diets have been applied to maintain the n-3 LC-PUFA contents in farmed fish. The present study validated the relevant efficacy.Takifugu rubripes). Two replacement strategies were used: a standard Graded Dietary Replacement of FO with BT (GDR strategy) and Alternate Feeding between FO- and BT-based Diets (AFD strategy). The positive and negative control diets were formulated with 6% FO (FO-C group) or BT (BT-C group) as the sole added lipid source. In the GDR strategy, three experimental diets were formulated, with 25, 50 and 75% of the added FO in the FO-C diet replaced with BT, named 25BT, 50BT and 75BT, respectively. In the AFD strategy, alternated feeding patterns between the FO-C and BT-C diet—namely, 1, 2 and 3 weeks with BT-C followed by 1 week feeding with FO-C —were applied. Each diet or feeding strategy was assigned to triplicate tanks. The results showed that dietary BT inclusion reduced the contents of long-chain polyunsaturated fatty acids (LC-PUFA) in both the muscle and liver (edible tissues for this species) of the experimental fish, and the liver displayed a more drastic decrease than the muscle. The LC-PUFA content linearly decreased with the decreasing dietary FO levels in the GDR strategy. However, in the AFD strategy, a linear relationship was not observed between the LC-PUFA content and the FO feeding duration. The 3BT-1FO treatment resulted in higher LC-PUFA content than 2BT-1FO. When comparing the two strategies with the same final FO administration level—namely, 50BT vs. 1BT-1FO, and in particular, 75BT vs. 3BT-1FO—the AFD strategy resulted in higher LC-PUFA contents in both the muscle and liver than the GDR strategy. In conclusion, when FO was replaced with BT in the diets, alternate feeding between FO- and BT-based diets resulted in a higher LC-PUFA content than the standard direct replacement. Three weeks of feeding with BT-C followed by one week of feeding with FO-C appeared to be a good alternate feeding pattern. This study provided a promising strategy of FO-sparing in fish farming when the LC-PUFA contents were maintained as high as possible.A 12-week feeding experiment was conducted to evaluate the effects of replacing fish oil (FO) with beef tallow (BT) on the fatty acid composition of farmed tiger puffer ( For human consumption, fish are the main source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), characterized by docosahexaenoic acid 22:6n-3, DHA) and eicosapentaenoic acid 20:5n-3, EPA). With the rapid expansion of aquaculture, farmed fish products have become a larger contributor to food fish intake than wild-caught fish products . Howevern-3, EPA.n-3, DHA Lateolabrax japonicus), cobia (Rachycentron canadum), Florida pompano (Trachinotus carolinus), totoaba , Atlantic salmon  and rainbow trout (Oncorhynchus mykiss), not only in terms of the LC-PUFA content [Among the alternative lipid sources, beef tallow (BT) has been reported to be a good substitute for FO in fish feeds. Beef tallow is rich in saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), which are preferentially oxidized for energy production in fish ,25. Bala content ,36,37,38 content ,40,41.Takifugu rubripes) is a traditional and important aquaculture species in Asia [Tiger puffer : 2 control groups; 3 treatments of Graded Dietary Replacement of FO with BT (GDR strategy); 3 treatments of Alternate Feeding between FO- and BT-based Diets (AFD strategy) . The posEnteromorpha proliferate in the near sea caused an instantaneous mortality . After that, we immediately switched the water source to deep-well saltwater. The remaining fish were in good health status in the following days (about 10 weeks). Therefore, the feeding trial continued to the end of week 12. The water quality parameters were monitored weekly: average temperature, 19–21 °C; salinity, 28–30; dissolved oxygen, >6.08 mg L−1; pH, 7.59–7.84.Juvenile tiger puffers  were purchased from Hongqi Aquaculture Co. Ltd. , and transported to Haiyang Aquaculture Co. Ltd. , where the feeding experiment was conducted. In order to prevent cannibalism, the fish teeth were cut shorter. The fish were fed a commercial diet for 14 days  during acclimation. After acclimatization, the fish were fasted for 24 h, weighted and randomly distributed into 24 tanks (200 L) at a stocking density of 35 fish per tank. Each feeding regime was assigned to triplicate tanks. Fish were hand-fed to apparent satiation 3 times a day . The tanks were cleaned daily by siphoning out the feces and residual feeds. In the first 2 weeks of the feeding trial, sand-filtered seawater was used. However, an outbreak of harmful algae v/v), the fish were individually counted and bulk weighed. After that, from each tank, two fish were randomly selected, and the body length and weight, as well as the viscera and liver weight, were recorded to calculate the condition factor (CF = body weight/body length3 × 100), hepatosomatic index (HSI (%) = wet liver weight/fish body weight × 100) and viscerosomatic index (VSI (%) = wet viscera weight/fish body weight × 100). Then, these two fish were used for the analysis of whole-body proximate composition. Four more randomly selected fish per tank were dissected and the tissue samples, including muscle, liver and intestine, were collected for subsequent potential use. All collected samples were immediately frozen with liquid nitrogen, and stored at -76 °C before use. All sampling protocols and fish handling practices of this study were reviewed and approved by the Animal Care and Use Committee of Yellow Sea Fisheries Research Institute.Sampling was conducted at the end of the feeding trial. Before sampling, the fish first were fasted for 24 h. After anesthetized with eugenol  were analyzed through gas chromatography . Briefly, fatty acids in lyophilized tissue samples were esterified with KOH-methanol and HCL-methanol in 72 °C water bath. Fatty acid methyl esters were extracted with hexane and then analyzed through gas chromatography using a flame ionization detector and a fused silica capillary column . The column temperature increase was programmed: 150 °C to 200 °C at 15 °C min-1; then from 200 °C to 250 °C at 2 °C min-1. The injector and detector temperatures were 250 °C. Fatty acid results were expressed as percentage of each fatty acid with respect to total fatty acids (% TFA).The measurement of the mitochondrial DNA (mtDNA) copy number in muscle and liver, as well as the hepatic expression of lipid metabolism genes, were conducted through qRT-PCR analysis.For the measurement of mtDNA, DNA in fish tissues was extracted using a TIANamp Genomic DNA Kit . For the measurement of lipid metabolism genes, total RNA in the liver was extracted using RNAiso Plus , Dalian, China) and reverse transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa), according to the user manual.The primers used are presented in -ΔΔCt method [The qRT-PCR system was based on 5 μL SYBR Green Pro Taq HS Premix II, 3.2 μL sterilized water, 1 μL cDNA template and 0.4 μL forward primer and reverse primer (10 μM). The thermal cycling conditions consisted of an initial denaturing step at 95 °C for 30 s, 40 cycles of “95 °C for 5 s, 57 °C for 30 s and 72 °C for 30 s”, and a final melting curve from 65 °C to 97 °C (6.4 °C increment/min). The relative gene expression levels were expressed according to the 2t method .The concentration of total cholesterol (TC), total triglyceride (TG), total bile acid (TBA), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum was measured using commercial kits supplied by the Nanjing Jiancheng Bioengineering Institute .p < 0.05.All data were analyzed using one-way analysis of variance (ANOVA) (SPSS 16.0 for Windows). Before analysis, all percentage data were arcsine transformed. Duncan’s multiple range test was used to detect significant differences between the means. A significant difference was accepted when Due to an early mortality in the feeding trial, the growth performance was no longer a focus of this study. In general, compared to the FO control group, the other diets resulted in no significant changes in final body weight .p < 0.05) lower than the other groups, with the exception of 75BT. In contrast, the FO replacement with BT affected the whole-body moisture content in an opposite way. The effects of the FO replacement with BT on the total lipid and moisture contents in the liver were similar to the trends described above. No significant difference was observed in the whole-body crude protein content and proximate composition of muscle among the different treatments.The final average fish body weight was 73.4 g. With respect to the whole-body proximate composition, the FO replacement with BT reduced the whole-body total lipid content, and the reduction was more severe in the GDR strategy than in the AFD strategy . Groups p < 0.05) lower HSI and VSI than the control groups, but the FO replacement with BT resulted in no significant (p > 0.05) changes in the CF compared to the control groups , in all of the tissues—but particularly the liver—the AFD strategy resulted in higher DHA and EPA contents but lower SFA and MUFA contents than the GDR strategy.p < 0.05) higher CYTB expression than the FO-C group, and the AFD strategy resulted in higher CYTB expression than the GDR strategy.In general, the expression of the two genes indicated that the mtDNA copy number was similarly different among the different feeding regimes . In the vlc, acox3 and acaa1 in the liver , Japanese seabass (Lateolabrax japonicus) and Atlantic salmon  [Sparidentex hasta) and large yellow croaker (Pseudosciaena crocea) [The total replacement of FO with BT significantly reduced the DHA content  in the muscle. However, a recent study on the same species showed that BT resulted in higher muscle DHA content than linseed oil, soybean oil, rapeseed oil and palm oil when FO was completely replaced . Similaro salar) ,39,52, a crocea) ,54.With respect to the partial replacement of FO with BT, the graded dietary replacement linearly decreased the muscle DHA content, but in the alternation feeding strategy, the muscle DHA content was not linearly correlated with the BT administration level. Studies have shown the existence of cyclical mechanisms relative to FA retention/utilization ,25. ThisMore importantly, the present study is the first to compare the efficacy of two different FO replacement strategies. The results showed that in both comparison sets with the same FO replacement levels—namely, 50BT vs. 1BT-1FO and 75BT vs. 3BT-1FO—the ADF strategy resulted in higher muscle DHA contents than the GDR strategy (the muscle DHA in 3BT-1FO is 108% that of 75BT). This could be related to the compensatory effects mentioned above. On the other hand, it could also be due to the fact that in the AFD strategy, before the final sampling, the fish were fed the FO-based diet for 1 week, which was equal to a miniature FO-finishing period. Nevertheless, if this cyclical miniature FO-finishing period works, it is meaningful in aquaculture practices.In the liver—which is also an edible tissue for this species—although, in general, similar results were observed, two main differences were observed. Firstly, compared to muscle, the hepatic EPA content was decreased more drastically by dietary BT inclusion. This was partly due to the fact that the liver is a lipid storage organ in tiger puffer , and thuMorone chrysops ♀ × M. saxatilis ♂), SFA were more strongly preferred for catabolism [It was hypothesized that mainly SFA and MUFA spared the n-3 LC-PUFA. The differences in the SFA and MUFA contents among the fish tissues of the different groups were indeed smaller than those among the diets. This was apparent for typical SFA and MUFA such as 14:0, 16:0, 16:1n-7 and 18:1n-9. SFA and MUFA are favorable substrates for β-oxidation in the energy production of fish ,60, and tabolism . This stIn order to evaluate the general energy supply status, the mitochondrial DNA copy number in the muscle and liver was assayed in the present study. The lack of differences in mitochondrial DNA copy number indicates a similar energy supply status among the groups, although the mitochondrial DNA copy number was not directly related to a certain fatty acid composition. Alternate feeding between FO- and BT-based diets even tended to increase the mitochondrial DNA copy number in the fish tissues.Gadus morhua), another species with high polar lipid contents in the muscle, do not have such a high 18:0 content in their muscle [In addition to SFA and MUFA, 18:2n-6 was also differently abundant between FO and BT. Previous studies have suggested that tiger puffer may have a low capacity for utilizing n-6 fatty acids . The higr muscle ,65,66,67r muscle ,69,70. TThe intestine is not an edible part. However, previous studies have shown that in tiger puffer, the intestine could be a lipid metabolism center, whereas the liver is more likely a pure lipid storage organ . In thisvlc, acox3 and acaa1 in the liver, but did not significantly affect the expression of the mitochondria fatty acid β-oxidation gene, cpt-1. Previous fish studies have indicated that peroxisomal β-oxidation accounts for a high proportion (30–50%) of total hepatic β-oxidation [lipc , which catalyzed the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins in the liver in the BT groups. In addition, the expression of lipogenic genes acacβ and fas was not affected by the dietary treatments, indicating that the dietary replacement of FO with BT has limited effects on lipogenesis.Nevertheless, although the changes in gene expression were relatively mild, some of the changes are still worth discussing. In particular, the inclusion of dietary BT up-regulated the expression of some peroxisomal fatty acid β-oxidation genes, including xidation ,72, but xidation . The predgat1, which is a key acyltransferase in triacylglycerol formation [mgll, which catalyzes the last step of triacylglycerol hydrolysis. This result indicates that in this pattern of alternation between FO- and BT-based diets, triacylglycerol may be better utilized. This was consistent with the superiority of this group in terms of the growth performance.Regarding the genes for glyceride biosynthesis and hydrolysis, compared to the FO-C group, the 3BT-1FO group showed significantly lower expression of ormation , but sigbasl and pl, which non-specifically hydrolyzes triacylglycerol completely to glycerol and free fatty acids, and hydrolyzes triacylglycerol specifically at sn-1,3, respectively [hmgcr, which is the rate-limiting enzyme of cholesterol biosynthesis, but down-regulated that of cyp7a1, which is the rate-limiting enzyme for bile acid synthesis from cholesterol [srebf1 and fxr were the most significantly regulated. These changes could be related to the changes in the gene expression of hmgcr and cyp7a1, respectively, considering the functions of these two transcription factors in the regulation of cholesterol and bile acid biosynthesis. Other gene expression results, such as the down-regulation of fabp10a and pparα2 and the up-regulation of lpl resulting from the dietary BT, were difficult to explain based on the current available information. The precise mechanisms involved need to be elucidated in future studies.High dietary BT levels down-regulated the expression of lipases ectively . This coectively . Regardilesterol . This coThe dietary inclusion of beef tallow reduced the LC-PUFA content in the muscle and liver of juvenile tiger puffer. However, the n-3 LC-PUFA sparing effect of SFA and MUFA in beef tallow can still be observed, particularly when alternate feeding between fish oil- and beef tallow-based diets was applied. Compared to direct fish oil replacement in the diets, the alternate feeding between fish oil- and beef tallow-based diets resulted in higher n-3 LC-PUFA contents in the fish. Three weeks of feeding with the beef tallow-based diet followed by one week of feeding with the fish oil-based diet seem to be the best feeding strategy to maximize the n-3 LC-PUFA deposition and reduce the reliance on fish oil.


The values of the comparative fit index and Tucker-Lewis index for the model of ECR-R-10 were between .903 and .985, whereas the root-mean-square error of approximation was between .082 and .036, indicating that the model fits were acceptable. The ECR-R-10 was related to the measurements with a similar construct; however, no difference in the magnitude of correlation was observed between ECR-R-18 and ECR-R-10. Known group validity was established. Measurement invariance was successfully established across different age and gender groups, although it was only partially achieved with respect to clinical status. The ECR-R-10 provided equal or superior psychometric properties to the ECR-R-18 across age groups and settings. As it is a briefer scale, the ECR-R-10 can be practically used in general and clinical samples to reduce the burden of assessment, especially with older adults. Further investigation is needed to test the scale's temporal stability.The experiences of close relationships-revised (ECR-R) is a widely used 36-item self-report measurement for measuring adult attachment. However, various short versions of the ECR-R have been developed and tested psychometrically. Given the cultural impact, a short version of the Thai ECR-R should be derived from the existing Thai version of the ECR-R. This study aimed to develop a 10-item version of the ECR-R that demonstrates comparable psychometric properties to the previous Thai version and the 18-item ECR-R. This study included four studies with a total of 1,322 participants. In study 1, 434 adults in a nonclinical setting were used for the development of the 10-item Thai ECR-R and tested in an independent sample. Studies 2, 3, and 4 were conducted on 312 adults in the clinical setting, 227 older adults in the nonclinical, and 123 older adults in clinical settings. The Cronbach alphas and corrected correlations between the ECR-R-18 and the ECR-R-10 in each study were calculated. Confirmatory factor analysis of the first-order two-factor solution model with fit statistics was examined with each sample. Correlations of the ECR-R-18 and the ECR-R-10 with other measurements were presented and compared. Known-group validity and measurement invariance test were also examined. The Cronbach alphas of the ECR-R-10 among all samples were acceptable, ranging between .77 and .85 for avoidance subscales and between .82 and .86 for anxiety subscales. The corrected correlation between the ECR-R-18 and ECR-R-10 was between .61 ( The reason for this increasing emphasis on attachment is that it has been found to impact individuals at multiple levels, such as personality, coping styles, relationships, and health. Insecure attachment has been identified as a predisposing factor for a range of mental, physical, and interpersonal difficulties, including anxiety, depression, personality disorders, chronic diseases, relationship issues, substance abuse, and internet addiction. Attachment-related concerns also affect the working alliance in psychotherapy and may serve as a predictive factor for the success of therapy. Alongside clinician-administered measures, numerous self-report questionnaires have been developed to assess attachment in practical settings. A recent review has identified over 20 self-report questionnaires that have been utilized for this purpose2. The original 323-item dataset was re-analyzed by Fraley and colleagues using item response theory to become a revised version with 36 items3.The Experiences in Close Relationships (ECR), a common self-report measurement used to assess attachment in adults, was developed by Brennan, Clark, and Shaver4. The ECR-R has been used and shown to be a significant predictor of mental health and psychiatric problems such as stress, anxiety, and social avoidance7.It is widely recognized that the use of questionnaires represents a valuable method for assessing the construct of attachment. Specifically, the anxiety and avoidance subscales have demonstrated a high level of validity in measuring this construct. Attachment-related anxiety refers to an individual's fear of being rejected by others, which is associated with a need for excessive attention and a tendency to exhibit intense stress reactions when a partner is unavailable or indifferent. On the other hand, attachment-related avoidance involves a tendency to reject dependency and interpersonal proximity, accompanied by self-absorption and a reluctance to disclose personal information. Individuals who receive high scores on one or both of these subscales are typically considered to have an insecure attachment style, while those who score low on both anxiety and avoidance subscales are generally regarded as having a secure attachment style3. It has been tested and found to be acceptable with many other languages such as Greece, Hungarian, and Italian, including Thai samples11. Since the ECR-R has 36 items, shorter versions of ECR-R were developed. Based on different cultural backgrounds12, the valid attachment items for each developed measurement are different, despite the fact that they designed to elicit the same meaningful construct . The shorter version of the ECR-R included a 12-item scale (English)13, a 18-item Thai version, 12-item German version14, a 16-item Czech version15, and 14-item Slovak version16. The items of the short Thai ECR-R is different from the 12-item questionnaire used in Wei’s study13, and from a 12-item version of German ECR-R14. For example, only two items are the same between the Thai and German versions: "I often worry that my partner will not want to stay with me." and "I worry that romantic partners won't care about me as much as I care about them." Both are from the anxiety subscale, whereas none of the items is the same between the Thai and German versions for the avoidance subscale. This discrepancy may underscore cultural differences that influence the validity of them. All shorter versions, even in different languages, have demonstrated an acceptable fit for the model.The ECR-R (Experiences in Close Relationships-Revised) was originally developed and tested for its psychometric properties using Anglo-Saxon populations25. Model misfit mainly resulted from one negative item , an item in avoidance subscale. This may be due to cultural influences. As found in related studies, Thai (or other Asian) respondents are more prone than western people to have difficulty responding negative items27. Therefore, a revision of the ECR-R-18 is needed. In addition, a revised ECR-R should be shorter when administered to older people. In summary, the 18-item Thai ECR-R demonstrates certain psychometric issues and is excessively lengthy.The 18-item Thai ECR-R (ECR-R-18) has been used in both clinical and nonclinical samples, with ages ranging from 18 to 95 years. The ECR-R-18 has been employed in several research studies to predict psychological problems such as loneliness, anxiety, depression, and suicidalityThe primary objective of this study was to assess the psychometric properties of the revised Thai version of the Experiences in Close Relationships scale across diverse samples, including young adults, older adults, and individuals from both nonclinical and clinical settings. We aimed to investigate whether the newly developed Thai ECR-R would demonstrate comparable or improved validity and reliability when compared to the previous 18-item version of the Thai ECR-R. Based on our hypothesis, we anticipated that the new Thai ECR-R would exhibit equivalent or superior psychometric properties in terms of validity and reliability compared to its predecessor. By examining a range of samples and settings, we aimed to obtain a comprehensive understanding of the revised scale's performance and its potential suitability for assessing attachment-related experiences in the Thai population.30. The study involved four samples from four studies comprising adults in both clinical and non-clinical settings, as well as older adults in both clinical and non-clinical settings.A secondary analysis was conducted on data from several projects that used the ECR-R-18 questionnaire, which is detailed elsewhere31. In line with this, we have the flexibility to choose between 8, 10, or 12 items, maintaining an equal number across each subscale. Although all three options are viable, opting for twelve items would offer a more comprehensive and robust measure compared to 10 or 8. However, after careful consideration, we have decided to strike a balance between the extremes and settle on 10 items for our study. These can be done simply by selecting the highest loading items for each subscale (avoidance and anxiety). Choosing items based on their loading values has several benefits, (1) construct validity: Items with high loadings are more likely to accurately represent and measure the intended construct, enhancing the validity of the scale. (2) reliability: Including items with high loadings increases the internal consistency and reliability of the scale. These items tend to contribute the most to the overall measurement precision of the construct. (3) efficiency: focusing on items with high loadings allows for the selection of a smaller subset of items that capture the essence of the construct, making the scale more concise and efficient. In addition, a new measurement should also be less burdensome, especially for older people. The 10-item ECR-R consists of five items for the anxiety subscale, and five items for the avoidance subscale.As per the recommended guidelines, it is advised to include a minimum of four items per scale  to ensure the reliability of measurementsStudy 2: 312 The ECR-R-10 was examined in adults in clinical settings.Study 3: The ECR-R-10 was examined in 227 non-clinical older adult sample.Study 4: The ECR-R-10 was examined in 123 clinical older adult sample.Study 1: 434 adults in a nonclinical sample was used to determine which 10 items should be used for the short version (ECR-R-10) using exploratory factor analysis. The rationale behind using a nonclinical sample for our study is rooted in its proximity to the general population. The original development of the ECR-R scale was based on data collected from a general population rather than clinical populations, although it has been found applicable to clinical samples as well. The five items with the highest factor loadings of each subscale were selected to compose the ECR-R-10. Then 226 university students were used to examine the construct validity of the ECR-R-10 using confirmatory factor analysis.The following measurements were used along with the ECR-R in each study.Rosenberg Self-Esteem Scale (RSES), a 4-point Likert scale. The Thai RSES revealed good internal consistency (α = 0.87). State -Trait Anxiety Inventory (STAI) scale, a trait -anxiety scale developed with a 20 -item instrument used to explore trait -anxiety. It is a 4-point scale. The Thai version of STAI used in this study showed good internal consistency (α = 0.89).Study 1, Perceived Stress Scale-10 (PSS-10), an instrument that measures the degree to which life events are perceived as being stressful. This is a 5-point Likert scale instrument. The Thai version of the PSS-10 has demonstrated good internal consistency . The Multi-dimensional Scale of Perceived Social Support (MSPSS), a 12-item, 7-Likert scale self-reporting tool that records how much social support respondents require. The Thai version has demonstrated an acceptable reliability (α = 0.90).Study 2, Revised UCLA Loneliness Scale, a 6-item questionnaire (RULS-6) and uses a 4-point Likert scale. The Thai version has demonstrated an acceptable reliability (α = 0.75). The Multi-dimensional Scale of Perceived Social Support (MSPSS), a 12-item, 7-Likert scale self-reporting tool which records how much social support respondents require. The Thai version has demonstrated an acceptable reliability (α = 0.92).Study 3 Perceived Stress Scale-10 (PSS-10), an instrument that measures the degree to which life events are perceived as being stressful. This is a 5-point Likert scale instrument. The Thai version of PSS-10 has demonstrated good internal consistency . The Multi-dimensional Scale of Perceived Social Support (MSPSS), a 12-item, 7-Likert scale self-reporting tool that records how much social support respondents require. The Thai version has demonstrated acceptable reliability (α = 0.90). We also used the Core Symptoms Index (CSI), an instrument measuring anxiety, depression, and somatization. It consists of 15 items, all of which are based on a 5-point Likert scale. The CSI revealed a good internal consistency (α = 0.91).Finally, Study 4 used the 32. Missing values were managed by replacing them with the series’ mean. Exploratory factor analysis was carried out using a method of principal axis factoring for extraction, and varimax with Kaiser Normalization for rotation method. Confirmatory factor analysis was applied to determine a two-factor solution model, using the maximum likelihood method with oblique rotation. Model fit was evaluated using several indices, including the Comparative Fit Index (CFI) ≥ 0.95, the Tucker-Lewis Index (TLI) ≥ 0.9, the root-mean-square error of approximation (RMSEA) ≤ 0.6, and the standardized root-mean-square residual (SRMR) ≤ 0.0833.Descriptive statistics were used for data screening; all items revealed skewness and kurtosis <  ± 2)34. Known group validity was determined by calculating the difference of ECR-R scores between nonclinical and clinical groups. Finally, internal consistency reliability was assessed by calculating the Cronbach's α coefficient, with a reliability of more than 0.70 considered acceptable. Test–retest reliability was performed using intraclass correlation in nonclinical sample. These analyses were conducted using IBM SPSS version 22 and AMOS package version 22.To determine convergent and discriminant validity, Pearson's correlation coefficients were calculated between the ECR-R and other measures. To account for spurious correlation in the evaluation of the short-form, the corrected correlation coefficients obtained by Levy’s method were used to calculate the correlation coefficients between ECR-R-18 and ECR-R1035, the following fit criteria of the factorial model were applied, CFI and TLI ≥ 0.90, SRMR < 0.05 and RMSEA ≤ 0.136. In multigroup CFA, Goodness-of-fit statistics were estimated for each model and for each model relative to the previous, less restricted, model. The ΔCFI and ΔRMSEA between the more and less constrained models were evaluated. The ΔCFI and △TLI larger than 0.01 and ΔRMSEA larger than 0.015 indicated a significant worsening of fit37. This analysis was performed with Mplus 8.10 version.To determine the invariance of the new ECR-R, we performed multigroup CFA (MGCFA) between sexes on each sample (adult and older adult), and among four groups using confirmatory factor analyses and measurement invariance analyses. Based on ByrneThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the Faculty of Medicine, Chiang Mai University .Informed consent was obtained from all individual participants included in the study.The respondents’ characteristics of each sample follow. Study 2: 276 adults in clinical setting , all diagnosed with depressive disorder. Study 3, 236 non-clinical older adult sample , and Study 4, 803 clinical older adult sample , 23.7% depressive disorder).p < 0.01) for avoidance and 0.75 (p < 0.01) for anxiety subscales , whereas only the anxiety subscale was significantly related to RSES  . The 10 items drawn from the 18 items are shown in Table es Table . AvoidanCFA results yielded factor loadings between 0.68 and 0.74 for the avoidance construct, 0.53 and 0.74 for the anxiety construct. A two-factor solution with the fit indices as follows, CFI = 0.969, TLI = 0.950, RMSEA = 0.056 , suggested that the two-factor solution model had an excellent fit to the data Fig.  , and the CFA results yielded factor loadings between 0.48 and 0.83 for the avoidance construct, and 0.59 and 0.78 for the anxiety construct. A two-factor solution with the fit indices as follows, CFI = 0.955, TLI = 0.939, RMSEA = 0.068 , and SRMR = 0.0677, suggesting that the two-factor solution model was a good fit for the data.p < 0.01) for avoidance and 0.759 (p < 0.01) for anxiety subscales , whereas only anxiety subscale was significantly related to PSS scores  , all diagnosed depressive disorder. Cronbach’s alpha of each subscale was 0.85 and 0.86 for avoidance and anxiety, respectively , suggesting that the two-factor solution model had a good fit to the data (Table p < 0.01) for avoidance and 0.786 (p < 0.01) for anxiety subscales , whereas only anxiety subscale was significantly related to RULS scores  . Cronbach’s alpha for each subscale was 0.81 and 0.83 for avoidance and anxiety, respectively , suggested that the two-factor solution model had a good fit to the data (Table p < 0.01) for avoidance and 0.772 (p < 0.01) for anxiety subscales (Table p < 0.05) . Cronbach’s alpha of each subscale was 0.77 and 0.82 for avoidance and anxiety, respectively , suggesting that the two-factor solution model was an excellent fit to the data .Table p < 0.001), indicating known-group validity.Table Table p < 0.0001) for anxiety, 0.50 (p < 0.0001) for avoidance. For the ECR-R-10, the intraclass coefficient was 0.79 (p < 0.0001) for anxiety, 0.76 (p < 0.0001) for avoidance.Test–retest reliability was performed using intraclass correlation in study 1. For the ECR-R-18, the intraclass coefficient was 0.70 .Table In general, the abbreviated Thai version of the ECR-R-10 demonstrates a significant correlation with the ECR-R-18 but offers superior psychometric properties. This can be attributed to the removal of a bad fit item, specifically item 1 . The 10-item version has been shown to possess construct validity within four distinct populations, including nonclinical and clinical adults, as well as nonclinical and clinical older adults. However, it is noteworthy that the model fit among older adults appears to be comparatively lower than in younger adults. In summary, the abbreviated Thai version of the ECR-R-10 has exhibited satisfactory reliability and validity for utilization in both age groups and various settings. Regarding construct validity, the data align well with a two-factor solution model, and concurrent validity as well as known-group validity have been supported. Furthermore, in addition to the observed acceptable to excellent levels of internal consistency as indicated by Cronbach's alphas, the nonclinical sample of the initial study demonstrated favorable results for test–retest reliability over a six-week timeframe.38, it is expected that the items of the ECR-R are different. Three out of five of the avoidance items and two out of five of the anxiety items are consistent with the Korean version, whereas four out of five (80%) of the Thai ECR-R-10 items are included in the 18-item Spanish version. This may be attributable to cultural differences in how respondents perceive avoidance and anxiety in relationships, as well as the characteristics of the sample. In terms of internal consistency, all shorter versions developed including the present study are good to excellent. Like the ECR-RD8 , the first order two-factor solution model in the current study fitted the data the best.When compared to other short versions of the ECR-R in English, Czech, Spanish, German, and Korean39.Unlike other studies that were mostly conducted using nonclinical samples, the present study was conducted in both clinical and nonclinical settings using the same set of items. Notably, item 1 , which was included in the 18-item Thai version, seems to have a poor fit to the model and is not included in other versions. The fact that this item does not sufficiently contribute to the construct when compared to other items makes it difficult to draw definitive conclusions. One possible hypothesis is that this variance results from the fact that this item is the only item that has different direction from the rest. In addition, it is the only item that is negatively wordedThe ECR-R-10 demonstrated measure invariance across sexes and age but not in different clinical status. This suggests that there are differences in the scaling or measurement units between the two groups, but the underlying structure or relationships between the observed variables and latent constructs are similar. The failure of scalar invariance implies that the measurement scales are not comparable between the nonclinical and clinical samples. The observed scores in the two groups might be influenced by factors specific to each group, such as differences in symptom severity, response biases, or other unexplored contextual factors related to the clinical condition.The findings of this study indicate that the ECR-R-10, in both nonclinical and clinical populations, demonstrated strong validity and reliability. Therefore, this shorter version of the questionnaire can be effectively utilized in a wide range of applications. Notably, the ECR-R-10 exhibited superior construct validity when compared to the longer ECR-R-18 version. Given its robust psychometric properties, the 10-item version is a viable and recommended alternative to the 18-item questionnaire. Researchers and practitioners can confidently use the ECR-R-10 as a reliable measure of attachment-related constructs while enjoying the benefits of its brevity and efficient administration.Despite the merits of brevity and wide use, the ECR-R-10 has some limitations. First, the sample used for analysis might not be representative of nonclinical adults because most participants were young adults, whereas the clinical sample used in this study was confined to psychiatric outpatient, mostly those with depressive disorders. It cannot represent all ranges of clinical samples. Secondly, it is important to note that the term 'clinical' used in this study was quite broad and encompassed participants with various diagnoses and levels of severity. This broad categorization might pose challenges when conducting measurement invariance testing. Therefore, it is recommended that future research endeavors employ a more specific and refined classification for clinical status and explore the possibility of developing group-specific measurement models. Thirdly, test–retest reliability has not been fully examined in all studies, and further investigation of this stability should be conducted in other populations, particularly in clinical settings. Lastly, it is worth noting that the present study utilized multiple instruments in addition to the ECR-R questionnaire. This inclusion of diverse instruments to validate the questionnaire across different populations and time frames may have influenced the study's results. Therefore, it is imperative to conduct replication studies to comprehensively examine the set of items used in the questionnaire. By replicating the study and utilizing consistent instruments, we can further validate the findings and ensure the robustness of the questionnaire's items.The ECR-R-10 provided sufficient psychometric properties across nonclinical and clinical adult and older adult cohorts. As it is brief, it can be administered to many age groups with less burden, especially older adults. Testing for the scale's stability, such as test–retest reliability, should be further investigated.Supplementary Information.


Bisphenol A type benzoxazine (Ba) monomers and 10--10- hydrogen-9- oxygen-10- phosphine-10- oxide (DOPO-HQ) were employed to prepare flame retardant and heat insulated polybenzoxazine (PBa) composite aerogels. The successful preparation of PBa composite aerogels was confirmed by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). The thermal degradation behavior and flame-retardant properties of the pristine PBa and PBa composite aerogels were investigated with thermogravimetric analysis (TGA) and cone calorimeter. The initial decomposition temperature of PBa decreased slightly after incorporating DOPO-HQ, increasing the char residue amount. The incorporation of 5% DOPO-HQ into PBa led to a decrease of 33.1% at the peak of the heat-release rate and a decrease of 58.7% in the TSP. The flame-retardant mechanism of PBa composite aerogels was investigated by SEM, Raman spectroscopy, and TGA coupled with infrared spectrometry (TG-FTIR). The aerogel has advantages such as a simple synthesis procedure, easy amplification, lightweight, low thermal conductivity, and good flame retardancy. It is well established that aerogels are solid materials with a three-dimensional nanoporous network structure, lightweight, high surface areas, and ultralow thermal conductivity. Aerogels have incomparable advantages in flame retardation, heat insulation, electromagnetic shielding, and other fields. Polybenzoxazine (PBa) is a kind of thermosetting resin with excellent mechanical strength and thermal stability, as well as nearly zero volume shrinkage and no volatiles releasing during the ring opening cross-linking process of benzoxazine (Ba) monomers ,3,4,5,6.The development, application, and problems of conventional flame-retardant methods were outlined in recent years . It is wCurrently, there is a search for more materials that are lighter in weight and perform better. Previously, ring-opening polymerization of benzoxazine in solvents led to gelation and the formation of 3D polymer networks ,17,18. TThe aerogel structure can be stabilized further, which has many applications in hydrophobicity and improvement of mechanical strength ,32. In aIn this study, multifunctional aerogels with high flame retardancy and low thermal conductivity are prepared through a sol-gel method using Ba as a reaction monomer, hydrochloric acid as a catalyst for ring-opening benzoxazine monomers, and DOPO-HQ as a flame retardant. DOPO-HQ effectively enhanced the flame-retardant properties of the PBa aerogels. This work provides cost-effective and environmentally friendly strategies for manufacturing flame-retardant polymer aerogels.−1 are attributed to the P-O-Ar, and the absorption peak near 1450 cm−1 corresponds to the P- Ar vibrations. It is observed that the absorption peaks of P-O-Ar and P- Ar are enhanced with the increase in DOPO-HQ content. In addition, all aerogels show broad O-H absorption peaks around 3420 cm−1, which is due to the ring opening of benzoxazine. The characteristic absorption peaks in the range of 1580–1700 cm−1 are due to the motion of the benzene ring caused by the C=C of the skeleton [The structural formula for DOPO-HQ(DQ) is shown in skeleton ,36,37. FPBa aerogels of various shapes and sizes can be obtained by sol-gel and atmospheric drying methods. The porous surface morphologies of PBa, PBa/DQ-1%, PBa/DQ-3%, and PBa/DQ-5% aerogels were investigated by SEM shown in 2 g−1, respectively. The pore size distribution map in The porous structure of the aerogel was further probed using nitrogen adsorption techniques. As shown in The thermal insulation performances of aerogels are shown in onset is the temperature at which the mass loss is 5%. The temperature Tmax is the temperature at which the maximum mass loss rate occurs. The amount of carbon residue is shown in onset of PBa/DQ-1% in the air decreased from 392 °C.The thermal degradation behaviors of PBa and PBa composite aerogels were analyzed through TGA . The onsonset decreased to 390 °C. Finally, for PBa/DQ-1%, Tonset decreased from 382 °C to 380 °C in the N2 atmosphere. Compared with PBa, the Tonset, and Tmax of PBa/DQ-5% composite aerogels decreased by 6 °C and 10 °C in air. Moreover, the Tonset and Tmax decreased in the N2 atmosphere by 11 °C and 4 °C. It is because DOPO-HQ contains a phosphorus group, which is more unstable than the carbon chain skeleton structure of the PBa matrix. This phosphorus group decomposes quickly and captures the water in the air to make acid. This process promotes the dehydration and carbonization of the matrix, which forms on the surface of the matrix. The density of the carbon layer prevents the exchange of heat and substances between the interior of the polymer and the surrounding environment [Similarly, for PBa, the Tironment ,44,45,462 [. The addition of DOPO-HQ can slow down the combustion reaction of the material, increase the carbon residue rate of the polymer, and improve its resistance to flames.From 2 . The add2O, and other substances. These substances are conducive to forming stable carbon residues, reducing heat and material transfer, and preventing further decomposition.PBa and its composite aerogels mainly occur between 300 and 500 °C. The less stable O=P-O bonds in DOPO-HQ can decrease the initial decomposition temperature of PBa and PBa composite aerogels. When temperatures exceed 300 °C, DOPO-HQ can produce phosphoric acid or polyphosphoric acid, HThe cone calorimeter is a fire test instrument capable of evaluating the combustion characteristics and potential fire safety of polymers. Cone calorimetry experiments were used to evaluate the combustion properties of PBa composite aerogels and high-temperature cured benzoxazine solid slab (PBr). All samples were cut to 100 mm × 100 mm × 3 mm in size for the CONE test. The heat release rate (HRR), total heat release rate (THR), carbon monoxide production (COP), and total smoke release rate (TSR) are shown in 2, which is 27.9% lower than that of PBa. The lower AvHRR value of DOPO-HQ in PBa composite aerogel can promote the formation of a dense protective layer on the material’s surface, reducing the possibility of a combustion reaction. In 2, which is 33.1% lower than that of PBa and 44.9% lower than that of PBr. The significant reduction in PHRR is mainly because the introduction of DOPO-HQ into the PBa composite aerogel promotes the formation of a cross-linked structure in the polymer matrix and a dense and compact carbon layer on its surface. That is why composite aerogel has better fire performance.Time to ignition (TTI) was used to assess the effect of DOPO-HQ on the flammability of composite aerogels. It can be seen from 2 to 17.1 MJ/m2, indicating that the addition of flame retardants could reduce the heat release rate of benzoxazine. The introduction of DOPO-HQ can create a physical barrier that helps reduce mass transfer and fire hazards during combustion. It is worth noting that smoke is more deadly than burning. The COP value can assess the toxicity released by the material during combustion. Compared with PBa, the COP value of the composite aerogel with 5% DOPO-HQ content was significantly reduced, as shown in Moreover, in the combustion reaction process, the smoke generation is mainly caused by incomplete combustion. In addition, the decrease in TSR and TSP values means that the addition of DOPO-HQ has a significant smoke suppression effect on the PBa composite aerogel. The PBa/DQ-5% results in a 59% reduction in TSP for PBa, reducing the risk of suffocation for trapped persons in the fire . TherefoSEM was performed to investigate the morphology of the residual carbon for pure PBa and PBa composite aerogels, as shown in 1–d1) shows that the structure of the surface is compact and continuous by PBa/DQ-5% compared to pure PBa. This structure can prevent the escape of small molecules of combustible gas and the heat transfer of the burning surface and the material under the carbon layer. 2–d2) shows that adding DOPO-HQ increases the internal porous structure. It can be seen from 2 and mass transfer. As shown in Furthermore, Raman spectroscopy was employed to measure the chemical structure of the char residue obtained from cone calorimetry tests. The degree of graphitization is the ratio ID/IG, reflecting the defect level or integrity of carbonaceous materials. When materials decompose, a higher graphitization of the char layer indicates a remarkably restrained performance on O−1, 2900–3000 cm−1, 2350 cm−1, and 1500–1750 cm−1 corresponding to H2O and NH3, alkane, carbon dioxide, and aromatic compounds, respectively. In 2 peak in the TG-FTIR spectrum of PBa/DQ-5% composite increased in 2 concentrations in the gas phase, thereby inhibiting combustion to some extent. In addition, the peaks in In order to study the role of D-fu in the gas phase during combustion, the pyrolysis products of PBa and PBa composite aerogels were analyzed by the TG-FTIR technique. Three-dimensional (3D) TG-FTIR spectra were recorded during the thermal decomposition process, as shown in Most importantly, the peaks can be attributed to the tensile vibrations of the P-O and P=O bonds, respectively f,h. ThesThe speculation on FR for the PBa/DOPO-5% composite is illustrated in Bisphenol A type benzoxazine (Ba) was provided by Sichuan Tiance Jucai Technology Co., Ltd. 10-10h-9-oxa-10-phospha-phenanthrene-10-oxide (DOPO-HQ) was purchased from Shanghai Xushuo Biotechnology Co. Ltd., . N, N-Dimethylamide (DMF), and ethanol were bought from Chengdu Kelong Chemical Reagent Factory . All chemicals were used as received without further purification.First, 5 g of bisphenol A benzoxazine and 40 g of DMF were added to a three-necked flask (250 mL). The mixture was stirred at room temperature to dissolve the benzoxazine monomer completely. After that, 1.25 g of concentrated hydrochloric acid was added and stirred for 60 min under ice bath conditions. After the solution turned from light to dark yellow, 0.05 g of DOPO-HQ (0.01 mol) was added and stirred until fully dissolved. The benzoxazine mixed solution was finally obtained.First, the mixed benzoxazine/DOPO-HQ was poured into the mold and put in a constant-temperature oven. It was aged at 50 °C for 72 h to solidify. After that, the wet gel was removed and soaked in absolute ethanol for 72 h. The absolute ethanol was replaced every 12 h to replace the DMF in the wet gel. After removing DMF, the wet gel was dried at normal temperature and pressure, and finally, a composite aerogel of PBa/DQ-1% was obtained. Similarly, PBa, PBa/DQ-3%, and PBa/DQ-5% aerogels were prepared according to the same experimental procedure, as shown in Polybenzoxazine panel (PBr) as a control sample was prepared through the following route. A total of 80 g of Ba was melted and poured into a glass mold and pre-cured at 180 °C/2 h and further reacted at 200 °C/2 h. The final material was marked as PBr. The thickness of PBr, PBa, and PBa/DQ samples was all around 3 mm.−1 range using a Nicolet 6700 spectrometer . The X-ray photoelectron spectrometer model used in this study was Nexsa from Thermo Scientific. The aerogel surface morphologies and carbon layers were analyzed with a scanning electron microscope (SEM) ZEISS EV0 MA15. Aerogel pore size distribution was assessed using a Mike ASAP2460, the adsorbed gas is nitrogen, degassed at 120 °C for 8 h. The thermal insulation properties of the aerogels were tested using a Hot Disk TPS 2500 S. TGA was performed using a DSC823 TGA/SDTA85/e from 40 °C to 800 °C at a ramp rate of 10 °C/min in nitrogen and air. The fire retardance performance of polymer materials were evaluated by cone calorimetry with ASTM E1354/ISO 5660. The samples were 100 mm × 100 mm × 3 mm in size, encapsulated in aluminum foil, and measured at a heat flow of 35 kw/m2. The structure of char residue from the cone calorimetry test was analyzed using Raman spectroscopy (Hrobia Xplra plus Raman) equipped with a laser wavelength of 785 nm. Thermogravimetric analysis coupled with Fourier transform infrared (TG-FTIR) was conducted with a Perkin-Elmer STA 6000 in nitrogen and air atmospheres with a heating rate of 10 °C/min from 40 °C to 800 °C.The pulverized aerogel surface functional groups were tested by FTIR spectroscopy in the 4000–500 cmVarious methods were employed to analyze the effect of DOPO-HQ on the micro-structure properties such as thermal degradation, flame retardance, and heat insulation. This work successfully fabricated PBa composite aerogels with excellent flame-retardant properties. PBa/DQ-5% aerogel has low thermal conductivity and good thermal insulation performance because of its uniform pore size. Compared with PBa aerogel, coke production increased at 700 °C and 800 °C with DOPO-HQ aerogel. In addition, cone calorimetry, Raman spectroscopy, and TG-IR tests confirmed that the addition of DOPO-HQ achieved aerogel flame retardancy and reduced the production of toxic gases and smoke. PBa/DQ aerogel has a potential application prospect in the field of high flame retardation requirements.